Reference no: EM133164401

NASC 1009 Introduction to Biosciences - University of South Australia

Chemistry Practical Experiment

Aims
• Qualitative analysis
• Use of chromatographic techniques
• Thin-layer chromatography (TLC) and column chromatography
• Separation of mixtures

Introduction

Chromatography, from the Greek words for colour and writing, is a term used to describe a variety of separation techniques. The components of a mixture to be separated are partitioned between two phases, one of which remains stationary while the other phase (mobile phase) percolates through the stationary phase, or over its surface. The mobile phase can be a liquid or a gas and the stationary phase can be a solid or a liquid - several combinations of stationary and mobile phases are thus possible.

PART A - THIN LAYER CHROMATOGRAPHY (TLC)

Procedure:
Capillaries with very small diameters are used as TLC spotters. You have been provided with TLC spotting pipettes to use to spot your samples onto the TLC plate.
1.) A clean spotting pipette will be needed for each sample, put them in a particular arrangement so that you do not mix up what has been spotted with what sample. If you get mixed up- get a fresh spotter or you will cause errors.

2.) Into the TLC developing jar add sufficient solvent (solvent A) to cover the bottom of the jar to about 0.5 cm. Close the lid on the jar lightly to allow a solvent atmosphere to form while preparing the TLC plate.

NOTE: when handling the TLC plates in the following steps please handle via the edges as fingerprints will interfere with the plate.

3.) Using one TLC. Take a piece of the silica gel-coated TLC sheet and lightly rule a pencil line across (approx. 1cm from the bottom of the plate, see Figure), taking care not to score through the absorbent layer (the silica). Then place small spots (1 mm diameter) of A, B, C and M only, on the line using the spotters (see Figure). Where A, B and C are the dichloromethane solutions of aspirin, paracetamol and caffeine. The spots need to be small so as to diminish spreading of the spots on development. Allow the spots to dry between each application (See TLC troubleshooting page in this booklet about problems with spotting on TLC).

4.) Then show a demonstrator your TLC to ensure that you have sufficient sample (will be viewed under UV light-UV lamps are in the balance room) before developing your TLC.

5.) Stand the TLC plate in the development jar, ensuring that the sampling line is above the level of the liquid in the beaker. Cover the beaker once more with the lid and watch closely as the solvent ascends up the TLC plate via capillary action.

Take care not to disturb the jar too much during this time.

6.) When the solvent has ascended to within 1.5 cm of the top of the plate (this should take approx. 20-25 minutes), mark the upper limit of the solvent front with a pencil, remove the plate, and allow to dry on benchtop (or in fumehood if needed).

7.) Examine the plate under a UV lamp (short wavelength, 254 nm) and mark the positions of the spots (carefully circle each spot you can see with a pencil). Sketch your chromatogram below clearly marking the positions of the spots and the solvent front. Each spot should be carefully outlined with a pencil.

8.) CARE: UV light may be harmful to unprotected eyes. Get help from a demonstrator if you need it -to analyse the developed plate under UV light.

9.) Now measure the distance between the ruled line and the solvent front and between the ruled line and the centre of each spot. Determine the Rf value of each of the reference drugs. Calculate Rf values (see over the page for help with calculating Rf values and/or ask a demonstrator for help) and identify the drug components present in the mixture.

10.) Report your results in your report book and then proceed to the second part of the practical session.

Procedure:

1.) Tear (or cut with scissors) some spinach leaves into strips and grind them to a paste using a mortar and pestle.

2.) Add a small amount (about 5 mL) of ethanol at a time, and stir/grind until the solution becomes dark green. As ethanol evaporates more may need to be added.
This is your solution of extracted pigments. Note: you may need to add more ethanol to your paste as it evaporates to make a liquid consistency.

3.) Filter the extract through a very small wad of cotton wool (or folded filter paper) in the neck of a small filter funnel into a small clean and dry test tube held in a clamp or a mini-test tube holder (see lockers). The stationary phase for the chromatography will be fine silica powder (found in the fume hood) held in a glass column.

4.) Take a glass Pasteur pipette (this is what will be your mini glass column) and use a
tiny wad of cotton wool to partially restrict the narrow capillary exit.

5.) Add fine silica powder into the pipette to make the column 5-6 cm long. Optional: wear face mask if concerned about silica. Then add a small top sand layer of 2-4mm.

6.) Clamp the pipette in the vertical (using a retort stand, boss head and clamp) and place a small beaker under it. This apparatus set-up can now be moved into a spare fume hood so there is minimal risk of inhalation of solvent as you run the column.

7.) Next, using a clean plastic Pasteur pipette place 2-4 drops of the (filtered) and hopefully very green plant extract on the top of the column of silica, allow 30-60 seconds for the material to adsorb onto the top of the column and to evaporate off and dry.

8.) Add the chromatographic solvent very gently so as not to disturb the surface of the silica with your sample extract on top (10% ethanol in petroleum ether - in the fume hood) to the top of the column, to fill the rest of the pipette.

9.) Keep carefully adding the solvent to the top of the column as it passes through the sample and down the column. NOTE: Add carefully or you will disturb the silica.

10.) Take note of the coloured bands that form as you add the chromatographic solvent and watch the separation that takes place. Draw a picture(s) of it over the page.
These are the purified chlorophylls and other extracts (see notes at the beginning of part B for more details).

11.) Try to identify the bands, make a note of the colours and the bands and show your demonstrator. You only need to develop the column until you see the separation.

12.) Once you have finished put any solvent waste in the container provided and your mini spinach column can go straight into the glass container in the fume hood (ask a demonstrator where this is).

13.) Pack up, tidy up and leave your bench/lockers clean and neat for the next group! Note: you may need to use detergent to clean up the mortar and pestle. Get your locker checked before leaving the lab as part of your lab participation.

Attachment:- Introduction to Biosciences.rar

Attachment:- practical report.rar